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Browsing by Subject "Confocal Microscopy"

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    Microfabrication Approaches for Understanding the Role of Laminin Output in Extracellular Matrix Composition in Chronic Obstructive Pulmonary Disease
    (2019-07) Boysen, Michael
    Chronic obstructive pulmonary disease (COPD) is an umbrella term used to indicate chronic bronchitis, emphysema, or a mixture of the two. Affecting over 10 million people and currently the 4th leading cause of death in the United States, COPD is prevalent amongst many aging individuals, with the severity of symptoms worsening over time1. To date, there is very little knowledge of COPD mechanisms and treatment methods. A decrease in laminin production, a key component of extra cellular matrices (ECMs), has been correlated with a decrease in lung function. We aimed to study the potential of lung ECMs modified to output increased laminin, namely lama3 and lama4, to reset older lung ECM to a younger state. To achieve this, we utilized modified muscular thin films (MTFs) with human lung fibroblasts (HLFs) that we called pulmonary thin films (PTFs). Stress in the cell monolayer of the PTF due to contraction, one key measure of pulmonary tissue function, causes the construct to bend. From this, the resulting contractile force can be derived due to known mechanical properties of PDMS, thickness of PTFs, and the radius of the curved beam upon contraction. First, we used microcontact printing of the modified ECMs on the PTFs. Then, after experiencing difficulty seeding on the substrate using microcontact printing, we employed a microfluidic deposition method with genipin to increase ECM adherence to the polydimethylsiloxane (PDMS) substrate. While we were able to successfully fabricate, seed HLFs, and experiment on PTFs using fibronectin with both microcontact printing and the deposition method, we were unable to seed cells with the correct line pattern using either method. These results pushed us to try a different method to evaluate the force output of HLFs on our modified ECMs – Traction force microscopy (TFM). TFM utilizes experimentally observed ECM displacements via a fluorescent microbead laden substrate to calculate traction force vectors generated at the surface of a cell. Furthermore, force production is a key measure not only of HLF function, but pulmonary tissue function as well. We found that the HLF came in two different morphology categories – spindle and non-spindle. When analyzing data of both morphologies collectively, little was found with regards to the ECMs influence on cell force output. However, when analyzing the morphologies separately, the spindle morphology followed the trend hypothesized – the increased laminin output in older ECMs increased the traction force output of HLFs causing them to be statistically different from their nonmodified, old control comparison. To this end, our data suggested that increased laminin output displayed the potential to return normal, young function to old lung ECM and may be key in future therapy methods for combatting ailments suffered by patients with COPD.